Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. Since Six1 does not have its own activation or repression domain, the Eya family of Six1 co-activator proteins is essential for Six1-mediated breast tumorigenesis and metastasis. We have developed an AlphaScreen based HTS assay targeting the Six1/Eya interaction. We propose to perform a large scale high throughput screening using the NIH MLPCN compounds to identify inhibitors of the Six1/Eya interaction. We plan to test these inhibitors for their potential as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and metastasis. These inhibitors can also be used as valuable chemical probes for studies involving the Six1/Eya interaction.